Process for the intracellular over-production of streptokinase using genetically engineered strain of E.coli

ABSTRACT

An improved process for the production of streptokinase using a genetically engineered strain of  Escherichia coli  which overproduces streptokinase intracellularly and more particularly, the overall process disclosed herein, concerns with an improvement in the fermentative production of streptokinase using an optimized growth medium mainly comprised of simple salts and trace-elements; thus, in principal, the present process constitutes an improved and more economical means for the production of streptokinase which may be useful in thrombolytic therapy.

FIELD OF THE INVENTION

The present invention disclosed herein, relates to an improved processfor the production of streptokinase using a genetically engineeredstrain of E. coli PSK4 deposited at the Microbial Type CultureCollection, at the Institute of Microbial Technology, Chandigarh, India,bearing Accession number MTCC 5120, which overproduces streptokinaseintracellularly. More particularly, the overall process disclosedherein, concerns with an improvement in the fermentative production ofstreptokinase using an optimized growth medium mainly comprised ofsimple salts and trace-elements. Thus, in principal, the present processconstitutes an improved and more economical means for the production ofstreptokinase which may be useful in thrombolytic therapy.

Background and Prior Art of the Invention

Streptokinase, an efficient activator of fibrinolysis, is routinelyutilized in clinical medicine for thrombolytic therapy for the treatmentof diverse circulatory disorders, e.g., pulmonary thrombo-embolism, deepvein thrombosis, and myocardial infarction. It exerts its fibrinolyticeffects through activation of an inert blood Zymogen, Plasminogen (PG),an active serine protease, Plasmin (PN), which attacks on fibrin todegrade it into soluble degradation products. It has been clearlydemonstrated, particularly in the case of myocardial infarction, that inthe actual management of the disease, streptokinase is as efficacious asits more expensive clot-dissolving alternatives, such as Urokinase (UK)and tissue-Plasminogen activator (tPA). Its utility in thrombolytictherapy is well established. Reference maybe made to the publications ofPaques, E. P., 1986, Haemostasis, Vol. 16, Suppl. 3, 21; ISIS-3 (ThirdInternational Study of Infarct Survival: A randomized comparison ofstreptokinase vs. tissue plasminogen activator vs. anistreplase and ofasprin plus heparin alone among 41,299 cases of suspected acutemyocardial infarction) Collaborative Group, 1992, Lancet 339,753.

Streptokinase is a single chain 47-kD protein, consisting of 414 aminoacid resides (reference may be made in the context of the biochemicalproperties of Streptokinase to the review article by Castellino, F. J.,1981, Chem Rev. 81, 431). It is naturally produced and secreted byvarious strains of hemolytic streptococci along with several otherunwanted toxic products, e.g. deoxyribonucleases, streptolysin orhyaluronidase and proteases, which makes the process of purifying thedesired protein difficult. On the other hand it has not yet beenpossible to obtain genetically improved strains from these hosts due tothe lack of a developed methodology for the gene transfer. Consideringits therapeutic applicability and clinical implications in thrombolytictherapy, attempts have been made in the past to search for analternative source of Streptokinase production through recombinantroutes. Reference is made to the publication of Malke and Ferretti,Proceedings of the National Academy of Sciences, USA, Vol. 81, p351,1984; Hagenson et al., 1989, Enzyme and Microbial Technology, Vol 11,650; Estrada et al., 1992, Biotechnology Vol 10, 1138; Reference mayalso be made to U.S. Pat. Nos. 5,296,366, 5,240,845, 4,764,469,2,043,953, Japanese Patent Number 2020828, European Patent No. 489201,and Cuban Patent No. 90. In the work reported in aforesaid publicationsand patents, the gene encoding for SK has been isolated from its naturalhost, Streptococcus and cloned into various heterologous hosts, e.g., E.Coli, Bacillus and yeasts.

In order to improve the yield of Streptokinase, in particular, the genewhich determines streptokinase C, A and G (References may be made to thepublications of Huang et al 1989, Molecular Microbiology Vol 2(3), 197;and Estrada et al, 1992, Biotechnology Vol 10, 1138;) were cloned andexpressed in E. coli as well as Streptococcus sanguis (Reference may bemade to the publication of Malke et al., 1984, Molecular and GeneralGenetics Vol 1%, 360). In both the cases, protein levels of 0.64 mg/land 40 μg/l, respectively, were obtained. In the case of E. coli, 94% ofthe protein recovered was in the periplasmic space and 6% in thecytosol, whereas, in S. sanguis all the enzyme was foundextracellularly. Moreover, many clones producing streptokinase were veryunstable owing to some lethal activity of the gene product or proteinsecretion. More recently, intracellular production of streptokinase hasbeen reported in E. Coli (Reference may be made to the publication ofXue-Wu Zhang et al, 1999, Enzyme and Microbial Technology, Vol 24,647)where 300-400 mg/ml SK protein has been obtained from one liter cellculture of E. coli using a rich and complex cell growth medium.

More often recombinant E. coli strain, developed for the overproductionof streptokinase, exhibited low cell density and biomass which affectedthe overall yield of the protein product. Production of recombinantstreptokinase in the methylotrophic yeast, Pichia pastoris, has beenattempted by Philips Petroleum Company (Reference may be made to thepublication of Hagenson et al., 1989, Enzyme and Microbial Technology,Vol 11, 650) and Cuban group (Reference may be made to the publicationof Estrada et al., 1992, Biotechnology Vol 10, 1138; and Cuban PatentNumber 90), where 1.8 mg/l and 1.0 g/l, respectively, of SK has beenobtained using continuous fermentation and rich growth medium comprisingof complex and expensive ingredients. Using a genetically engineeredstrain of E. coli 645 mg/l of SK production (Reference may be made tothe publication of Xue-Wu Zhang et al, 1999, Enzyme and MicrobialTechnology, Vol. 24, 647) has been reported under fermentative conditionusing a rich growth medium.

OBJECT OF THE INVENTION

Thus the prime objective of the present invention is to develop animproved and economical process for the production of streptokinaseusing a genetically engineered strain of E. coli which produces thisprotein intracellularly.

Yet another objective of the invention is to prepare a piece ofDeoxyribonucleic Acid (DNA) carrying complete genetic information forthe production of streptokinase inside the cell of recombinant E. coliobtained as above and to prepare an expression plasmid DNA (pSK4)carrying the genetic information for the production of Streptokinase ina suitable E. coli host. In this plasmid pSK4, p denotes plasmid and SKdenotes streptokinase. It has been deposited in the Microbial TypeCulture Collection at the Institute of Microbial Technology, under theAccession number MTCC 5120.

Yet another objective of this is to design a process for obtaining largequantity of cell biomass of genetically engineered E. coli PSK4(Accession No. MTCC 5120) using fermentative processes in order toimprove the overall production of streptokinase and to recover largeamounts of streptokinase during subsequent downstream processing.

SUMMARY OF THE INVENTION

The present process pertains to the development of an improved andeconomical process for the production of streptokinase using agenetically engineered strain of E. coli PSK4 bearing InternationalAccession number MTCC 5120 carrying a replicative plasmid vector,capable of producing streptokinase intracellularly in high yields.Streptokinase so obtained exhibits biological properties, e.g., clotdissolution and plasminogen activation characteristics, similar tonatural streptokinase. Further a fermentation process for the high-celldensity cell culture of this recombinant E. coli strain has beendeveloped utilizing an inexpensive growth medium composition, whichresults in a higher cell biomass yielding higher recovery ofstreptokinase from the cell extract. Following these steps, high-levelof streptokinase can be obtained from the cell culture of E. coli(Accession No. MTCC 5120) which is biologically active and may be usefulfor therapeutic purposes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved process for the production ofintracellular streptokinase using a genetically engineered strain of E.coli (deposited at the Microbial Type Culture Collection at theInstitute of Microbial Technology, Sector 39-A, Chandigarh—160 036,India under Accession No. MTCC 5120) overproducing streptokinaseintracellularly following an optimized batch and fed-batch fermentationregime using simple and inexpensive medium components. The processcomprises of:

-   1. Preparation of a piece of DNA carrying genetic information for    the production of streptokinase through, recombinant processes or    through a synthetic approach following known procedures.-   2. Integration of the above piece of DNA on a suitable replicable    plasmid vector through known recombinant processes to prepare a    replicable expression plasmid vector, capable of producing    streptokinase in a suitable host under appropriate conditions.-   3. Introduction of plasmid DNA prepared at step 2, into an    appropriate host cell such as such as E. coli, B. subtilis, or Yeast-   4. Adaptation of the host cell prepared at step 3 into a growth    medium of defined composition consisting of basal salts, trace    elements and carbon source.-   5. Adjustment of aeration in the range 0% to 100% dissolved oxygen,    agitation in the range 50 to 1000 revolutions per minute and pH in    the range 5 to 8.-   6. Addition of antibiotic such as Kanamycin, Ampicillin and the    like, in the range 1 to 1000 microgram per milliliter in the    fermenter-   7. Addition of seed culture of the host cell in the range 0.1% to    10% carrying the desired expression plasmid into the fermentation    medium-   8. Cultivating in the fermenter for a period ranging from 6 to 24    hours.-   9. Optionally feeding additional nutrients to the fermentation    medium at time intervals ranging from 15 minutes to 90 minutes and    at rates ranging from 100 mL per hour to 1000 mL per hour to enhance    biomass yields-   10. Addition of the inducer (IPTG) in the range 0.01 mM to 10 mM,    after sufficient cell biomass is attained.-   11. Harvesting of cell culture broth by conventional centrifugation    and/or microfiltration.-   12. Lysis/breakage of E. coli cells through chemical lysis,    sonication, bead mill or through French Pressure cell-   13. Isolation and resolubilization of streptokinase inclusion bodies    from fermentation broth-   14. Isolation and separation of streptokinase protein following    conventional chromatographic steps.

In yet another embodiment of the present invention, wherein the overallprocess developed and disclosed herein is primarily based on the use ofa genetically engineered strain of E. coli carrying a plasmid DNAencoding for the production of streptokinase for the high levelfermentative production of streptokinase level using defined growthmedium and physiological parameters such as aeration, pH, inductionconditions, nutrient feed etc. Large scale production of streptokinaseusing recombinant strains of E. coli (Accession No. MTCC 5120) has beendone mainly using complex growth medium for the recovery of largequantities of streptokinase. Using a specifically designed expressionplasmid, intracellular production of SK has been obtained underfermentation condition which yielded about 200-330 mg SK/L cell culture.

In still another embodiment of the present invention, wherein a piece ofDNA carrying genetic information for the production of streptokinase hasbeen retrieved from the recombinant plasmid construct, pJKD-21, carryingan open-reading frame cassette for the production of streptokinase.

In still another embodiment of the present invention, wherein theexpression plasmid was constructed by preparation of a piece of DNAobtained as above, and carrying the genetic information for theproduction of streptokinase through known recombinant or syntheticprocesses and integrated on to a suitable replicable plasmid like pET-9Aagain through known recombinant techniques to prepare a replicableexpression plasmid vector.

In still another embodiment of the present invention, wherein the saidplasmid expression vector was then introduced into an appropriate hostlike E. coli or B. subtilis or yeast using known transformationtechniques and adapted on to a synthetic medium comprising of a solecarbon source, basal salts and trace elements whose composition is asfollows: glycerol as carbon source in the range 10-35 g/L, Potassiumphosphate in the range 5-20 g/L, Ammonium phosphate in the range 2-6g/L. Magnesium sulphate in the range 1-3 g/L, Citric acid in the range1-2 g/L, EDTA in the range 5-10 mg/L, Cobalt chloride in the range 2-4mg/L, Manganese chloride in the range 10-20 mg/L, Copper chloride in therange 0.5-2.5 mg/L, Boric acid in the range 2-5 mg/L, Sodium molybdatein the range 1-5 mg/L, Zinc acetate in the range 5-25 mg/L, Ferricchloride in the range 50-100 mg/L, and Thiamine hydrochloride in therange 4-8 mg/L.

In still another embodiment of the present invention, wherein thisadapted culture was then cultured in the fermenter at a pH range of 5-8,aeration range of 0% to 100% dissolved oxygen, and agitation range of 50to 1000 revolutions per minute on the above synthetic medium.

In still another embodiment of the present invention, wherein 0.1% to10% of the seed inoculum also prepared using this synthetic medium wasinoculated into the fermenter containing sterile synthetic medium.

In still another embodiment of the present invention, wherein antibioticsuch as kanamycin, ampicillin and the like was added in the range 1 to1000 microgram per milliliter to the seed medium and fermetation mediumprior to the addition of culture organism. After 6 to 24 hours ofgrowth, inducer isopropyl-p D thiogalactopyranoside (IPTG) in the range0.01 mM to 10 mM was added to induce the culture to producestreptokinase.

In still another embodiment of the present invention, whereinfermentation was further carried out for a period ranging from 4 to 10hours and biomass harvested by-conventional centrifugation ormicro-filtration.

In still another embodiment of the present invention, wherein thebiomass thus obtained was lysed using known techniques of cell lyses andstreptokinase inclusion bodies harvested.

In still another embodiment of the present invention, wherein theharvested inclusion bodies of streptokinase were resolubilized in Ureaor Guanidine hydrochloride buffers and refolded and purified to obtainenzymatically active and pure streptokinase. In a preferred embodiment,1.3 kb amplified DNA was joined on suitable expression Plasmid vectors,such as pKK-233-2, pET-9A, Trc-99 etc (ranging from 30-50 copy/cell),preferably pET-9A, which was digested with restriction enzymes such asNde I, BamHI and the like. In another preferred embodiment, E. colistrains, BL 21DE3, or JM 105, preferably BL 21DE3, have been utilizedfor carrying plasmid vectors linked with Streptokinase encoding DNA.

In yet another preferred embodiment the trace elements and carbon sourcewere sterilized separately and added to the sterile fermenter containingthe basal salt solution. In yet another preferred embodiment additionalnutrient feed was added to the fermenter to enhance biomass. A furtherdescription of the invention is given in examples below, which shouldnot however be construed to limit the scope of the present invention.

EXAMPLE 1 Preparation of a Replicative Plasmid (pSK4) Expressing ofSignal-Peptide Free Streptokinase for the Intracellular Production ofStreptokinase in E. Coli (Accession No. MTCC 5120)

The source of DNA for retrieving mature Streptokinase encoding gene,lacking its native signal peptide sequences, was plasmid pJKD-21. 3 μgof plasmid pJKD-21 was digested with 5-10 units each of NdeI and BamHIrestriction enzymes using known techniques and incubated at 37° C. for 8hrs. Reaction was stopped by heating the reaction mixture at 80° C. for10 minutes and this digested sample was electrophoresed on 1% agarosegel for 1 h at 150 mV. 1.3 kb DNA fragment liberated from the plasmidpJKD-21 was excised from the gel and purified using gene-clean kit. In aparallel reaction, 1 μg of plasmid pET9a (commercially obtained from NewEngland Biolabs) was also digested with 5 and 10 units of NdeI andBamHI, respectively and Incubated at 37° C. for 8-10 hrs. This digestwas mixed with 0.5 μg of 1.3 kb DNA fragment of plasmid pJKD-21 in atotal volume of 50 μl. This DNA mixture was precipitated at −20° C. inthe presence of 100 μl of absolute ethanol and 5 μl of sodium acetate.DNA precipitate was dissolved in 17 μl of water and 3-4 units of T4DNAligase was added along with its complementary buffer (obtainedcommercially from New England Biolabs, USA). This reaction mix wasincubated at 16° C. for overnight and transformed in E. coli BL21DE3through known transformation procedure. Transformed cells were plated onLB (Luria-Broth) plates carrying 20 μg/ml Kanamycin antibiotic andincubated at 37° C. for overnight. 10 individual colonies were pickedout in 10 ml LB medium carrying 20 μg of kanamycin and incubated at 37°C. for 4-5 h till the cell OD reached to 0.5, after that 0.01 mM IPTGwas added and cells were further incubated for overnight. The cells werepelleted by centrifugation and analyzed on 10% SDS-PAGE. The coloniesexhibited an intense 47 kD protein band which was not present in thecontrol cells which were transformed with the control plasmid pET9a. Theselected clone was designated as pSK4 and deposited in the MicrobialType Culture Collection, Institute of Microbial Technology, Chandigarh,under the Accession number MTCC 5120. This was subjected to fermentationto realize the production level of Streptokinase and its biologicalcharacterization.

EXAMPLE 2 Adaptation of the Recombinant E. Coli to a Synthetic Medium

E. coli (Accession No. MTCC 5120 growing on complex media like LuriaBroth (LB) was adapted to synthetic medium comprising of glycerol ascarbon source 25 g/L, Potassium phosphate 13 g/L, Ammonium phosphate 4g/L, Magnesium sulphate 1.2 g/L, Citric acid 1.7 g/L, EDTA 8.4 mg/L, andtrace element solution (1 mL-10 mL) containing: trace quantities ofCobalt chloride, Manganese chloride, Copper chloride, Boric acid, Sodiummolybdate, Zinc acetate, Ferric chloride, and Thiamine hydrochloride.100 μl of LB grown culture in the early stationary phase of growth wasinoculated into a 100 mL shake flask containing 10 mL of pre-warmedsynthetic medium containing 50 μg/mL Kanamycin and incubated at 37° C.at 100 RPM in a rotary shaker for 8 hours. 100 μL of this culture wasinoculated into another 100 mL shake flask containing 10 mL ofpre-warmed synthetic medium containing 50 μg/μl Kanamycin and incubatedat 37° C. at 100 RPM in a rotary shaker for 8 hours. 1 ml of thisculture was further inoculated into a 500 mL shake flask containing 50mL of pre-warmed synthetic medium containing 50 μg/μl Kanamycin andincubated for 8 hours. 500 μl aliquots of this adapted culture was mixedwith equal volume of 50% glycerol in 1 ml vials and stored at −70° C. asGlycerol Stocks.

EXAMPLE 3 Preparation of Seed Culture of Recombinant E. Coli forFermentation

For each fermentation one vial of Glycerol Stock as prepared in Example2 was inoculated into a 500 mL shake flask containing 50 mL ofpre-warmed synthetic medium synthetic medium comprising of glycerol ascarbon source 25 g/L, Potassium phosphate 13 g/L, Ammonium phosphate 4g/L, Magnesium sulphate 1.2 g/L, Citric acid 1.7 g/L, EDTA 8.4 mg/L, andtrace element solution (1 mL-10 mL) containing trace quantities of:Cobalt chloride, Manganese chloride, Copper chloride, Boric acid, Sodiummolybdate, Zinc acetate, Ferric chloride, and Thiamine hydrochloride.Kanamycin 50 μg/μl was added and incubated for 10 hours and was used asthe seed inoculum for all fermentations.

EXAMPLE 4 Batch Fermentation for the Production of RecombinantStreptokinase

2.0 L of basal salt medium comprising of Potassium phosphate 13 g/L,Ammonium phosphate 4 g/L, Magnesium sulphate 1.2 g/L, Citric acid 1.7g/L, EDTA 8.4 mg/L, were sterilized in a 3 L Chemap fermenter. Glycerol(10-35 g/L) preferably 30 g/L, and trace element solution (1 mL-10 mL)containing trace quantities of: Cobalt chloride, Manganese chloride,Copper chloride, Boric acid. Sodium molybdate, Zinc acetate, Ferricchloride, and Thiamine hydrochloride were all sterilized separately andadded to the cooled fermenter and the final volume made up to 2.45 Lwith sterile distilled water. pH was adjusted to 7.4 using 5N Sodiumhydroxide. 50 ug/uL of filter sterilized Kanamycin was added prior tothe addition of seed inoculum.

50 mL of seed inoculum as prepared in Example 3 was added to make thevolume finally to 2.5 L. The main cultivation was carried out at 37° C.for a period of 12 hrs. pH in the fermenter was maintained at 7.4 byusing aqueous ammonia and HCl. Fermenter RPM of 700 and an initialaeration rate of 2.5 L/min was set. RPM was slowly increased to 900 tomaintain a minimum dissolved oxygen concentration of 10% air saturation.The fermentation was carried out for 10 hours after which the fermenterwas induced with 0.2 mM IPTG when a biomass concentration of 7 g DCW(dry cell weight)/L was achieved in the fermenter. Fermentation wascontinued for another 4 hours and biomass harvested for streptokinaserecovery.

EXAMPLE 5 Fed Batch Fermentation for the Production of RecombinantStreptokinase

2.0 L of basal salt medium comprising of Potassium phosphate 13 g/L,Ammonium phosphate 4 g/L, Magnesium sulphate 1.2 g/L, Citric acid 1.7g/L, EDTA 8.4 mg/L, were sterilized in a 3 L Chemap fermenter. Glycerol(10-35 g/L) preferably 30 g/L, and trace element solution (1 mL-10 mL)containing trace quantities of: Cobalt chloride, Manganese chloride,Copper chloride. Boric acid, Sodium molybdate, Zinc acetate, Ferricchloride, and Thiamine hydrochloride were all sterilized separately andadded to the cooled fermenter and the final volume made up to 2.45 Lwith sterile distilled water. pH was adjusted to 7.4 using Sodiumhydroxide. 50 μg/μl of filter sterilized Kanamycin was added prior tothe addition of seed inoculum 50 mL of seed inoculum as prepared inExample 3 was added to make the volume finally to 2.4 L. The maincultivation was carried out at 37° C. pH in the fermenter was maintainedat 7.4 by using aqueous ammonia and HCl. Fermenter RPM of 700 and aninitial aeration rate of 2.5 L/min was set. RPM was slowly increased to900 to maintain a minimum dissolved oxygen concentration of 10% airsaturation. The fermentation was carried out for 10 hours after which 50ml of fresh feed consisting of Glycerol 795 g/L, Magnesium sulphate 20g/L, EDTA 13 mg/L, Cobalt chloride 4 mg/L, Manganese chloride 23.5 mg/L,Copper chloride 2.5 mg/L, Boric acid 5 mg/L, Sodium molybdate 4 mg/L,Zinc acetate 16 mg/L, and Ferric chloride 40 mg/L was added. Thefermentation was further carried out for 40 minutes and another 50 ml offresh feed containing Glycerol 795 g/L, Magnesium sulphate 20 g/L, EDTA13 mg/L, Cobalt chloride 4 mg/L, Manganese chloride 23.5 mg/L, Copperchloride 2.5 mg/L, Boric acid 5 mg/L, Sodium molybdate 4 mg/L, Zincacetate 16 mg/L, and Ferric chloride 40 mg/L was added. The fermenterwas induced with 1 mM IPTG when a biomass concentration of 18 g DCW (drycell weight)/L was achieved in the fermenter. Fermentation was continuedfor another 4 hours and biomass harvested for streptokinase.

EXAMPLE 6 Isolation and Resolubilization of Inclusion Bodies fromFermentation Broth

Fermentation broth was centrifuged at 5000 g for 15 minutes at 4 C, torecover biomass. The cells were washed in a buffer containing (i) 10 mMTris HCl, pH 8, (ii) 10 mM EDTA, and (iii) 100 mM Sodium chloride andre-centrifuged. The cell pellet was re-suspended in the buffercontaining (i) 10 mM Tris HCl, pH 8, (ii) 10 mM EDTA, (iii) 100 mMSodium chloride, (iv) 1 mM Phenyl methyl sulfonyl fluride. Lysozymepreferably 0.64% was added to the above solution and stirred for 3 hoursat 4° C. prior to sonication for 30 minutes. The sonicated solution wascentrifuged at 5000 g to obtain streptokinase inclusion bodies.

8 M Urea or 6 M Guanidine HCl was added to the above isolated inclusionbodies and stirred for 24 hours and centrifuged at 5000 g. Thesupernatant was diluted 100 fold using a buffer containing 0.05 mM Trisat a pH of 7.5 and dialyzed to obtain active recombinant streptokinase.

1. A genetically engineered strain of E. coli PSK4 deposited in theMicrobial Type Culture Collection at the Institute of MicrobialTechnology, Chandigarh, India, bearing Accession number MTCC
 5120. 2. Agenetically engineered strain as claimed in claim 1, wherein the abovesaid strain is having the following typical characteristics: a) itcarries a recombinant 4.3 kilobase plasmid pSK4, b) this recombinantplasmid has a streptokinase encoding gene fragment, and c) it carrieskanamycin resistant marker which provide Kanamycin resistance.
 3. Anovel medium composition useful for the production of streptokinase,said composition comprising Potassium phosphate of concentration rangingbetween 5-20 g/L, Ammonium phosphate of concentration ranging between2-6 g/L, Magnesium sulphate of concentration ranging between 1-3 g/L,Citric acid of concentration ranging between 1-2 g/L, EDTA ofconcentration ranging between 5-10 mg/L, Cobalt chloride ofconcentration ranging between 2-4 mg/L, Magnesium chloride ofconcentration ranging between 10-20 mg/L, Copper chloride ofconcentration ranging between 0.5-2.5 mg/L, Boric acid of concentrationranging between 2-5 mg/L, Sodium molybdate of concentration rangingbetween 1-5 mg/L, Zinc acetate of concentration ranging between 5-25mg/L, Ferric chloride of concentration ranging between 50-100 mg/L,Thiamine hydrochloride of concentration ranging between 4-8 mg/L; andoptionally Casein hydrolyste of concentration ranging between 0.5-1.0%,and Yeast extract of concentration ranging between 0.2-0.5%.
 4. Aprocess for the production of intracellular streptokinase usinggenetically modified E. coli PSK4 (MTCC 5120), said process comprisessteps of: a. adapting the genetically modified strain of E. coli havingAccession No. MTCC 5120 in a growth medium having carbon source, basalsalts and trace metals to prepare seed culture; b. adding the seedculture of step (a) to a fermentation medium and fermenting the same fora period of 8 to 12 hors; c. adding an inducer to the fermentationmedium of step (b) and fermenting the medium for another 3 to 5 hours toobtain the inclusion bodies, and d. harvesting, lysing andresolubilizing the inclusion bodies of step (c) to obtain thestreptokinase.
 5. A process as claimed in claim 4, wherein in step (a)the growth medium comprises Potassium phosphate in the range of 5-20g/L, Ammonium phosphate in the range of 2-6 g/L, Magnesium sulphate inthe range of 1-3 g/L, Citric acid in the range of 1-2 g/L, EDTA sin therange of 5-10 mg/L, glycerol in the range of 10-35 g/L, 1-10 ml traceelement solution containing trace quantities of Cobalt chloride,Magnesium chloride, Copper chloride, Boric acid, Sodium molybdate, Zincacetate, Ferric chloride, Thiamine hydrochloride and optionally Caseinhydrolyste 0.5-1.0%, and Yeast extract 0.2-0.5%.
 6. A process as claimedin claim 4, wherein in the step (b) the fermentation medium comprisesPotassium phosphate in the range of 5-20 g/L, Ammonium phosphate in therange of 2-6 g/L, Magnesium sulphate in the range of 1-3 g/L, Citricacid in the range of 1-2 g/l, EDTA in the range of 5-10 mg/L, glycerolin the range of 10-35 g/L, Cobalt chloride in the range of 2-4 mg/L,Magnesium chloride in the range of 10-20 mg/L, Copper chloride in therange of 0.5-2.5 mg/L, Boric acid in the range of 2-5 mg/L, Sodiummolybdate in the range of 1-5 mg/L, Zinc acetate in the range of 5-25mg/L, Ferric chloride in the range of 50-100 mg/L, Thiaminehydrochloride in the range of 4-8 mg/L; and optionally Casein hydrolyste0.5-1.0%, and Yeast extract 0.2-0.5%.
 7. A process as claimed in claim4, wherein in step (b) the fermentation is carried out in a optimizedbatch process or a fed batch process.
 8. A process as claimed in claim4, wherein in step (b) and (c) the fermentation time is in the range of8-17 hrs.
 9. A process as claimed in claim 4, wherein in step (b) the pHof the medium is in the range of 6-8.
 10. A process as claimed in claim4, wherein in step (b) the temperature is in the range of 30-40° C. 11.A process as claimed in claim 4, wherein in the step (b) the temperatureis in the range of 35-39° C.
 12. A process as claimed in claim 4,wherein in the step (b) the RPM is in the range of about 700 to
 900. 13.A process as claimed in claim 4, wherein in the step (b) the dissolvedoxygen concentration is in the range of about 5-20%.
 14. A process asclaimed in claim 14, wherein the dissolved oxygen concentration is about10%.
 15. A process as claimed in claim 7, wherein the process in the fedbatch fermentation, nutrients are added at one or more intermediatestages of fermentation.
 16. A process as claimed in claim 15, whereinnutrients are added at two intermediate stages of fermentation with atime of about 40 to 60 minutes between them.
 17. A process as claimed inclaim 16, wherein the nutrient comprising glycerol in the range of600-900 g/L, EDTA in the range of 10-15 mg/L, Cobalt chloride in therange of 3-5 mg/L, Magnesium chloride in the range of 15-30 mg/L, Copperchloride in the range of 2-5 mg/L, Boric acid in the range of 3-8 mg/L,Sodium molybdate in the range of 2-7 mg/L, Zinc acetate in the range of5-25 mg/L, Ferric chloride in the range of 50-100 mg/L.
 18. A process asclaimed in claim 4, wherein the step (c) the inducer is isopropylthiogalactoside (IPTG).
 19. A process as claimed in claim 18, whereinthe inducer is added in the range of 0.01 mM to 10 mM when the cellbiomass of 7-18 g dry cell weight/L is attained.
 20. A process asclaimed in claim 4, wherein in step (d) the harvesting the biomass iscarried out by centrifugation and/or microfiltration.
 21. A process asclaimed in claim 4, wherein in step (d) the lysis of inclusion bodies iscarried out by chemical lysis or sonication or bead mill or throughfrench pressure cell.